Molecular basis for the high degree of antigenic cross-reactivity between hepatitis B virus capsids (HBcAg) and dimeric capsid-related protein (HBeAg): insights into the enigmatic nature of the e-antigen

Abstract

The hepatitis B virus core gene codes for two closely related antigens: a 21-kDa protein that forms dimers that assemble as multimegadalton capsids, and a 17-kDa protein that also forms dimers but that do not assemble. The proteins, respectively referred to as core antigen (HBcAg) and e-antigen (HBeAg), share a sequence of 149 residues but have different amino- and carboxy-termini. Their structural and serological relationship has long been unclear. With insights gained from recent structural studies on immune complexes of the capsids, the relationship was reassessed using recombinant forms of the antigens and a panel of monoclonal antibodies (mAbs) commonly believed to discriminate between core and e-antigen. Surface plasmon resonance (SPR) was used to measure the affinities, in contrast to previous studies that used more error-prone and less sensitive plate-type assays. Four of the six mAbs did not discriminate between core and e-antigen, nor did they discriminate between e-antigen and dimers of dissociated core antigen capsids. One mAb (3120) was specific for assembled capsids and one (e6) was specific for unassembled dimers. Epitope valency of the e-antigen was also studied, using a sandwich SPR assay where e-antigen was captured with one mAb and probed with a second. The e-antigen is often considered to be a monomeric protein on the basis of monovalent reactivity with antibody pairs specific for either an alpha or beta epitope (in a prior nomenclature for e-antigen specificity). This model, however, is incorrect, because recombinant e-antigen is a stable dimer and its apparent monovalency is due to steric blockage. This was proven by the formation of a 2:1 Fab e6-e-antigen complex. These results suggest new approaches for the isolation of the authentic e-antigen, its biological assay, and its stabilization as an immune complex for structural studies.

Fig. 1

Schematic representation of the recombinant HBV proteins used in this study. The three capsid-related proteins (Cp) correspond to those listed in Table 1 and in greater detail in Fig. 2. Cp183 is expressed in E. coli as nucleic acid-filled capsids (Cp183_c) with a mass of ∼6 MDa (represented by the blue fenestrated structure). Such capsids are very stable and cannot be dissociated into subunits without protein denaturation. In Cp149 the arginine rich carboxy-terminal domain has been deleted and when this protein is expressed in E. coli it forms empty capsids (Cp149_c) with masses of 3 and 4 MDa (represented by a grey fenestrated structure). These capsids can be reversibly dissociated into dimeric subunits with a mass of ∼35 kDa (grey object). The red line indicates an intermolecular disulfide bond [C61-C61]. The Cp(-10)149 or e-antigen is expressed in E. coli in a form that is neither assembled capsid nor an aggregated inclusion body-type protein. The protein can be extracted with 2 - 3 M urea at pH 9.5 to give soluble folded dimers. Purified dimers can be induced to form capsids (white fenestrated structure). The reversibility of this has not been fully explored. The red lines indicate intramolecular disulfide bonds [C(-7) – C61]. See Figs. S1 and S2 for analytical data on these various proteins.

Fig. 2

Locations of the epitopes on HBV capsids and subunits. The cartoon represents three adjacent dimeric subunits on a capsid with the protruding spikes corresponding to the four-helix bundles. The epitopes for Mabs 88, 312, 842, 3105, 9c8, and F11A4 are all located on the apices of the spikes whereas the epitope for Mab 3120 is located between the spikes and involves residues from two adjacent subunits.; ; ; ; The two copies of the Mab e6 epitope are located near the C-termini of the dimer and are accessible only on free subunits. The locations of the epitopes for Mabs 904 and 905 are uncertain, but epitope 904 may be on the spike apex and 905 near the C-terminus (see text). The positions of the three carboxy-terminal mutations employed to map the e6 epitope, and which affect capsid assembly, are also indicated.

Fig. 3

Typical sensograms illustrating the binding of antigen to Mab 3105. Antigen was allowed to bind to immobilized antibody. Duplicate, and in some cases triplicate, injections of antigen were done. The concentrations refer to the concentration of antigen, in terms of dimers. The kinetics and affinities of Cp149_c (A) and Cp149_d (B) for Mab 3105 are similar, indicating that the epitope is not affected significantly by the assembly state of the antigen. This is in keeping with its known location on the apex of the spike(s).

Fig. 4

Immune complexes visualized by electron microscopy. Cp(-10)149_d were mixed with antibodies in solution and visualized in negative stain. (A) Mab alone, (B) Dimer + Mab 904, (C) Dimer + Mab 905. Outlines and an interpretative diagram are shown in each case. The formation of the circular complexes in (C) suggests the presence of two copies of the 905 epitope per Cp(-10)149_d molecule. Very similar complexes were observed when Mab e6 was employed (not shown). Bar = 200 Å.

Fig. 5

Characterization of Cp149_d:Fab e6 and Cp(-10)149_d:Fab e6 immune complexes. Analysis of the complexes by (a) gel filtration chromatography, (b) analytical ultracentrifugation, and (c) reducing SDS-PAGE indicates a 1:2 molar ratio in both cases.